Fig. 7. Localization of centromere- and checkpoint-related proteins in Nuf2- and Hec1-deficient cells. (A) Localization analysis of Hec1-GFP in Nuf2-deficient cells and Nuf2-GFP in Hec1-deficient cells. In control cells (–tet), Hec1-GFP (green) and Nuf2-GFP (green) signals are localized at the centrosome (G1/S phase) and centromere (mitosis). After addition of tet, both signals are reduced. DNA is counterstained with DAPI (blue). The panel on the right shows a western blot analysis with a mixture of anti-Nuf2 and anti-Hec1 antibodies of Nuf2- and Hec1-deficient cells. The levels of Hec1 in Nuf2-deficient cells and of Nuf2 in Hec1-deficient cells are reduced. (B) Immunofluorescence analysis with antibodies against CENP-A, -C and -H of prometaphase chromosomes in Nuf2- and Hec1-deficient cells after addition of tet. (C) Localization of checkpoint proteins in Nuf2- and Hec1-deficient cells. Both control cells (-tet) have strong Mad2-DsRed signals. After addition of tet, the Mad2-DsRed signals are diffuse in both mutants. Both mutants were also stained with anti-BubR1 (red) in the presence or absence of tet. BubR1 signals are present on the centromeres of cells in mitotic arrest caused by Nuf2 or Hec1 depletion. (D) Immunofluorescence analysis with antibody against -tubulin of Nuf2- and Hec1-deficient cells.